Ex vivo μCT images of the digits were obtained using a Bruker SkyScan 1,172 scanner (Bruker, Kontich, Belgium) at a pixel size of 4 µm with 0.2 rotation angle and five frame averaging using a custom 0.25 mm aluminum filter. The X-ray source used was 50 kV, 201 μA, and 10 W, as described previously (Tower et al., 2022 (
link)). All samples were reconstructed using NRecon with smoothing correction disabled, a beam hardening correction of 24%, and a dynamic range of 0.00–0.339. Reconstructed digits were exported as 8-bit BMP output files, rotated transaxially in DataViewer, and binarized and 3D analyzed in CTAn. Global thresholds were used for all young mice (8-week old) data sets with minimum threshold value set to 0 and maximum threshold value of 67 and a global threshold for aged mice (18-month old) data sets with minimum threshold value set to 0 and maximum threshold value of 79. For analysis of hypomineralized tissue at day 14 we used CTAn (Bruker, Kontich, Belgium, RRID:
SCR_021338) and the Binary Selection preview window to identify newly regenerated bone. Hypomineralized tissue was isolated by identifying mineralized areas below 67 (the 6-month old mouse threshold for bone in the digit) in the grayscale data stack. To exclude mineralized bone the Binary Selection preview window was used to set a global threshold with a lower bound of 35 and an upper bound of 67. The Morphological Operations plugin was used to remove the partial volume effect in the 3D binarized image using Opening, Round Kernel, and Radius of 1. The volume of hypomineralized tissue was quantified using 3D analysis. The taper of the digit morphology was quantified as previously described (Tower et al., 2022 (
link)). Briefly, we identified the start of newly regenerated bone using CTAn and measuring the area of the newly regenerated bone from the P3 cortical bone stump to the distal tip. The bone area was recorded and graphically represented along the length of the newly regenerated bone for control and OAA treated samples (Tower et al., 2022 (
link)). Bone mineral density (BMD) was calculated as previously described (Hoffseth et al., 2021a (
link)). Briefly, we calibrated attenuated X-ray data values from digit data sets to known mineral density standards of 0.25 and 0.75 mg calcium hydroxyapatite (CaHA) known as “phantoms” to determine the density of CaHA g/cm
-3 in mineralized tissue (Hoffseth et al., 2021a (
link)).
